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1.
J Biol Chem ; 293(14): 5210-5219, 2018 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-29475945

RESUMO

Electron transfer in all living organisms critically relies on formation of complexes between the proteins involved. The function of these complexes requires specificity of the interaction to allow for selective electron transfer but also a fast turnover of the complex, and they are therefore often transient in nature, making them challenging to study. Here, using small-angle neutron scattering with contrast matching with deuterated protein, we report the solution structure of the electron transfer complex between cytochrome P450 reductase (CPR) and its electron transfer partner cytochrome c This is the first reported solution structure of a complex between CPR and an electron transfer partner. The structure shows that the interprotein interface includes residues from both the FMN- and FAD-binding domains of CPR. In addition, the FMN is close to the heme of cytochrome c but distant from the FAD, indicating that domain movement is required between the electron transfer steps in the catalytic cycle of CPR. In summary, our results reveal key details of the CPR catalytic mechanism, including interactions of two domains of the reductase with cytochrome c and motions of these domains relative to one another. These findings shed light on interprotein electron transfer in this system and illustrate a powerful approach for studying solution structures of protein-protein complexes.


Assuntos
Citocromos c/química , NADPH-Ferri-Hemoproteína Redutase/química , NADPH-Ferri-Hemoproteína Redutase/ultraestrutura , Citocromos c/ultraestrutura , Transporte de Elétrons , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Cinética , NADP/metabolismo , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Difração de Nêutrons/métodos , Nêutrons , Oxirredução , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Relação Estrutura-Atividade , Termodinâmica
2.
Proc Natl Acad Sci U S A ; 114(7): 1474-1479, 2017 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-28087691

RESUMO

Imaging single proteins has been a long-standing ambition for advancing various fields in natural science, as for instance structural biology, biophysics, and molecular nanotechnology. In particular, revealing the distinct conformations of an individual protein is of utmost importance. Here, we show the imaging of individual proteins and protein complexes by low-energy electron holography. Samples of individual proteins and protein complexes on ultraclean freestanding graphene were prepared by soft-landing electrospray ion beam deposition, which allows chemical- and conformational-specific selection and gentle deposition. Low-energy electrons do not induce radiation damage, which enables acquiring subnanometer resolution images of individual proteins (cytochrome C and BSA) as well as of protein complexes (hemoglobin), which are not the result of an averaging process.


Assuntos
Holografia/métodos , Proteínas/ultraestrutura , Imagem Individual de Molécula/métodos , Animais , Bovinos , Citocromos c/ultraestrutura , Elétrons , Grafite , Hemoglobinas/ultraestrutura , Holografia/instrumentação , Soroalbumina Bovina/ultraestrutura , Imagem Individual de Molécula/instrumentação , Espectrometria de Massas por Ionização por Electrospray/métodos , Eletricidade Estática , Vácuo
3.
Biochem Biophys Res Commun ; 469(4): 978-84, 2016 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-26718409

RESUMO

Redox-dependent changes in the structure and dynamics of human cytochrome c (Cyt c) were investigated by solution NMR. We found significant structural changes in several regions, including residues 23-28 (loop 3), which were further corroborated by chemical shift differences between the reduced and oxidized states of Cyt c. These differences are essential for discriminating redox states in Cyt c by cytochrome c oxidase (CcO) during electron transfer reactions. Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments identified that the region around His33 undergoes conformational exchanges on the µs-ms timescale, indicating significant redox-dependent structural changes. Because His33 is not part of the interaction site for CcO, our data suggest that the dynamic properties of the region, which is far from the interaction site for CcO, contribute to conformational changes during electron transfer to CcO.


Assuntos
Citocromos c/química , Citocromos c/ultraestrutura , Oxigênio/química , Sítios de Ligação , Ativação Enzimática , Humanos , Cinética , Oxirredução , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade
4.
Int J Biol Macromol ; 58: 104-12, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23541553

RESUMO

Cytochrome c (cyt c) exists as a partially unfolded intermediate at 45 mM gallic acid (GA) possessing disrupted secondary structure, altered Trp environment and high ANS binding. Increasing the concentration of either GA or ferulic acid (FA) up to 50 mM results in cyt c aggregation as confirmed by shift in Congo red, increase thioflavin T, decrease ANS and Trp fluorescence. SEM confirmed the formation of fibrils and amorphous aggregates of cyt c in presence of 50 mM FA and GA respectively. Single cell gel electrophoresis establishes very less probability of this noble protein to cause misfolding and aggregation-prone diseases.


Assuntos
Antioxidantes/química , Ácidos Cumáricos/química , Citocromos c/química , Ácido Gálico/química , Animais , Benzotiazóis , Citocromos c/ultraestrutura , Corantes Fluorescentes/química , Cavalos , Microscopia de Fluorescência , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Multimerização Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Deficiências na Proteostase , Análise de Célula Única , Tiazóis/química , Triptofano/química
5.
Arch Biochem Biophys ; 528(1): 67-71, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-22985801

RESUMO

Tuning of both hydrophobic and electrostatic interactions is thought to be important for the initial nucleation and stability of protein aggregates that self-assemble to produce amyloid fibrils. Importance of a critical balance of these two interactions has indeed been determined under various solution conditions of fibrillation, the acidic pH, in particular. To find out if fibrillar protein structures could be obtained under extreme alkaline conditions, cytochrome c was allowed to fibrillate in 0.1 N NaOH at 50 or 60 °C. Fibers do grow in alkali, but the fibrillation process depends little on the ionic strength of the solution. Illustrative fibril morphology readily obtained even in the absence of solvent cations poses the question as to how the severity of electrostatic repulsions is overcome to initiate aggregation. It appears that intermolecular hydrophobic collapse is so overwhelming that electrostatic repulsions are subdued, and the negative charges on protein molecules are relocated in a way conducive to fiber growth. This proposal seems consistent with computer simulation studies indicating central role of hydrophobic interactions. Morphologically, branched fibrils characterized by a wide distribution of diameter are assembled by winding two or more protofibrils. The results should guide selection of model parameters in theoretical studies of fibrillation.


Assuntos
Amiloide/química , Citocromos c/química , Amiloide/metabolismo , Amiloide/ultraestrutura , Animais , Citocromos c/metabolismo , Citocromos c/ultraestrutura , Cavalos , Temperatura Alta , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Concentração Osmolar , Conformação Proteica , Desnaturação Proteica , Hidróxido de Sódio/metabolismo , Eletricidade Estática
6.
J Biochem ; 152(6): 521-9, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22923742

RESUMO

We have previously shown that polymerization of cytochrome c (cyt c) occurs by successively domain swapping its C-terminal α-helix in the presence of ethanol. However, the factors that govern the conversion process of monomers to domain-swapped oligomers remain unknown. We found that oligomeric cyt c is produced in the presence of ethanol and the oligomers precipitate due to low solubility. The optical absorption spectra revealed that in the presence of 30-40% ethanol, the Met-heme coordination in cyt c is dissociated. However, according to circular dichroism and small-angle X-ray scattering measurements, the α-helical structure of cyt c is maintained in solution with a little perturbation and the radius of gyration increases slightly but without dissociation of the C-terminal α-helix from the rest of the protein by the addition of ethanol. Solid-state nuclear magnetic resonance spectra showed that oligomeric cyt c in the precipitate also retains most of its α-helical structure. In the transmission electron microscopic image of the precipitate obtained by the addition of ethanol to cyt c, spherical particles with diameters of about 3 nm were detected. These results indicate that oligomeric cyt c forms through a state with the Met80 region locally unfolded, while maintaining the secondary structure, possibly an open monomer.


Assuntos
Citocromos c/química , Etanol/química , Animais , Precipitação Química , Cromatografia em Gel , Dicroísmo Circular , Citocromos c/isolamento & purificação , Citocromos c/ultraestrutura , Heme/química , Cavalos , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica de Transmissão , Oxirredução , Tamanho da Partícula , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Desdobramento de Proteína , Solubilidade
7.
Colloids Surf B Biointerfaces ; 82(1): 111-7, 2011 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-20832261

RESUMO

Cytochrome c (cyt c) is a small soluble protein from the intermembrane space of mitochondria. This protein is essential because it transfers electrons between two membrane complexes of the respiratory chain. In fact, during this transfer, the positively charged amino-acid residues surrounding the heme in the protein structure allow the cyt c to interact properly with the anionic part of other molecules: mainly the cardiolipin-rich membrane of mitochondria and respiratory complexes. We have previously shown that besides its interaction with anionic lipids, the cyt c is also able to cross neutral lipid membranes. In this work, with the help of AFM and punch-through experiments, we have measured the force required to penetrate the membrane in the fluid and in the gel phases with or without cyt c molecules. In the presence of cyt c molecules, the structures generated by the interaction with the protein were considerably weakened, which led to the desorption of the fluid bilayer and to a considerable loss of cohesion of the gel phase. These results show the usefulness of punch-through experiments in determining the changes of membrane properties in the presence of external agents.


Assuntos
Citocromos c/metabolismo , Membranas Artificiais , Microscopia de Força Atômica/métodos , Análise Espectral/métodos , 1,2-Dipalmitoilfosfatidilcolina/química , Animais , Fenômenos Biomecânicos , Citocromos c/ultraestrutura , Géis , Cavalos , Íons , Bicamadas Lipídicas/metabolismo , Fluidez de Membrana , Modelos Biológicos , Nanopartículas/química , Fosfatidilcolinas/química
8.
Biosens Bioelectron ; 24(8): 2618-24, 2009 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-19233635

RESUMO

Molecularly imprinted polymers (MIPs) are synthetic receptors obtained by polymerization using molecular templates. We have synthesized MIP films (co-polymers of acrylamide and different acrylic acid-based cross-linkers) with specific binding sites for cytochrome c, which were imprinted in the bulk or in the surface. The binding specificity of the polymers was studied at the macroscale by equilibrium binding experiments with fluorescein-labeled cytochrome c. Imprinting factors of up to 4.1 were obtained that were a function of the cross-linker used and the degree of cross-linking. We have then employed, for the first time, AFM force spectroscopy to directly measure the force of interaction of the protein with the synthetic receptor sites obtained by molecular imprinting. The polymer surfaces were scanned with AFM cantilevers carrying covalently attached cytochrome c molecules, giving raise to specific binding events with binding forces between 85 and 95 pN. Control cantilevers without cytochrome c or with covalently attached bovine serum albumin, as well as non-imprinted control polymers, did not yield specific binding events. We believe that these results demonstrate the great potential of force spectroscopy for the characterization of molecularly imprinted polymers.


Assuntos
Citocromos c/química , Citocromos c/ultraestrutura , Microscopia de Força Atômica/métodos , Técnicas de Sonda Molecular , Mapeamento de Interação de Proteínas/métodos , Ligação Proteica , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Estresse Mecânico
9.
Langmuir ; 24(16): 8779-84, 2008 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-18642859

RESUMO

We report a study of the electrostatic layer-by-layer self-assembly of electroactive polyelectrolyte multilayers incorporating the redox protein cytochrome c (cyt c) combined with recrystallization of the bacterial cell wall surface layer from Bacillus sphaericus CCM 2177 SbpA (S-layer). The polyelectrolyte multilayer assembly was prepared on flat gold electrodes with a nanometer-scale roughness that allowed monitoring of the film formation throughout all the assembly stages by atomic force microscopy measurements in liquid with respect to topography and forces. The deposition of alternating layers of sulfonated polyaniline and cyt c was carried out by adsorption from the corresponding solutions on a cyt c monolayer electrode. The electroactivity of cyt c within the assembly was confirmed by cyclic voltammetry. We showed that the surface properties of the electrode terminating layer change after each adsorption step accordingly. We also found that S-layer recrystallization on the top of the multilayer film was feasible while electroactivity of cyt c within a polyelectrolyte matrix was partially maintained. This approach offers a new strategy to design a biocompatible and permselective outer envelope of a polyelectrolyte multilayer, promising sensor applications.


Assuntos
Citocromos c/química , Eletrólitos/química , Animais , Cristalização , Citocromos c/metabolismo , Citocromos c/ultraestrutura , Cavalos , Microscopia de Força Atômica
10.
Biophys J ; 94(12): 4828-36, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18326641

RESUMO

Equilibrium unfolding behaviors of cytochrome c and lysozyme induced by the presence of urea (0-10 M) as well as changes in temperature (295-363 K) or pH (1.8-7) are examined via small-angle x-ray scattering and spectroscopic techniques, including circular dichroism and optical absorption. Denaturant and temperature effects are incorporated into the free energy expression for a general multigroup unfolding process. Results indicate that there are at least four unfolding groups in the temperature-, urea-, or pH-induced unfolding of cytochrome c: two of these are related to the prosthetic heme group, and the other two correspond, respectively, to the unfolding of alpha-helices and global changes in protein morphology that are largely unaccounted for by the first two groups. In contrast, the unfolding of lysozyme approximately follows a simple one-group process. A modified mean-field Ising model is adopted for a coherent description of the unfolding behaviors observed. Thermodynamic parameters extracted from simple denaturing processes, on the basis of the Ising model, can closely predict unfolding behaviors of the proteins in compounded denaturing environments.


Assuntos
Cristalografia/métodos , Citocromos c/química , Citocromos c/ultraestrutura , Modelos Químicos , Modelos Moleculares , Muramidase/química , Muramidase/ultraestrutura , Simulação por Computador , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína
11.
Biophys J ; 94(10): 4066-77, 2008 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-18227133

RESUMO

The alternative low-spin states of Fe(3+) and Fe(2+) cytochrome c induced by SDS or AOT/hexane reverse micelles exhibited the heme group in a less rhombic symmetry and were characterized by electron paramagnetic resonance, UV-visible, CD, magnetic CD, fluorescence, and Raman resonance. Consistent with the replacement of Met(80) by another strong field ligand at the sixth heme iron coordination position, Fe(3+) ALSScytc exhibited 1-nm Soret band blue shift and epsilon enhancement accompanied by disappearance of the 695-nm charge transfer band. The Raman resonance, CD, and magnetic CD spectra of Fe(3+) and Fe(2+) ALSScytc exhibited significant changes suggestive of alterations in the heme iron microenvironment and conformation and should not be assigned to unfold because the Trp(59) fluorescence remained quenched by the neighboring heme group. ALSScytc was obtained with His(33) and His(26) carboxyethoxylated horse cytochrome c and with tuna cytochrome c (His(33) replaced by Asn) pointing out Lys(79) as the probable heme iron ligand. Fe(3+) ALSScytc retained the capacity to cleave tert-butylhydroperoxide and to be reduced by dithiothreitol and diphenylacetaldehyde but not by ascorbate. Compatible with a more open heme crevice, ALSScytc exhibited a redox potential approximately 200 mV lower than the wild-type protein (+220 mV) and was more susceptible to the attack of free radicals.


Assuntos
Citocromos c/metabolismo , Citocromos c/ultraestrutura , Cavalos/metabolismo , Ferro/química , Modelos Químicos , Miocárdio/enzimologia , Análise Espectral/métodos , Animais , Simulação por Computador , Modelos Moleculares , Conformação Proteica , Marcadores de Spin
12.
Anal Bioanal Chem ; 390(5): 1253-60, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18157669

RESUMO

In this paper we report the first application of multivariate data analysis techniques to force spectrometry measurement sets to enable the physicochemical assignment of spatially ordered multi-component systems. Principal component analysis (PCA) and hierarchical clustering techniques were used to reveal hidden chemical information within force-distance curves generated by high spatial resolution force microscopy. Two experimental samples were analyzed: (i) a two-component system of cytochrome c proteins on a mica surface, and (ii) a three-component system of avidin protein islands positioned on a gold and glass surface. PCA and hierarchical clustering techniques were used to discriminate the different components of the two-component system, whereas hierarchical clustering was found to be superior for the three-component system. Results were in good agreement with the topography and prior knowledge of the surface patterns. This research represents a formative step towards the combination of force spectrometry with chemometric tools for the high resolution physicochemical investigation of complex biochemical systems.


Assuntos
Microscopia de Força Atômica/métodos , Adesividade , Análise por Conglomerados , Citocromos c/ultraestrutura
13.
Langmuir ; 23(17): 8659-62, 2007 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-17629308

RESUMO

In this work, surface-enhanced Raman scattering (SERS) was applied to probe the orientation of cytochrome c (Cyt-c) on gold nanohole arrays functionalized with self-assembled monolayers (SAMs) of alkane thiols with positively (-NH2) and negatively (-COOH) charged terminal groups. Square grid gold nanohole arrays with a nanohole diameter of 270 nm and a grating of 350 nm were fabricated by electron beam lithography (EBL) and were used as the SERS substrates. The SERS intensities of the nontotally symmetric mode (B(1g) mode nu(11)) and the totally symmetric mode (A(1g) mode nu(4)) and their ratios were used to determine the orientation of Cyt-c on surfaces. The results indicate that the heme group is close and perpendicular to the negatively charged surface but is far from and oriented at an angle to the positively charged surface. Cyt-c has a random or more flat orientation on the bare Au nanoholes surface.


Assuntos
Citocromos c/química , Citocromos c/ultraestrutura , Ouro/química , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Análise Espectral Raman/métodos , Microscopia Eletrônica de Varredura , Modelos Moleculares , Estrutura Molecular , Propriedades de Superfície
14.
Biochem Biophys Res Commun ; 360(1): 194-8, 2007 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-17583674

RESUMO

Multiheme c-type cytochromes from members of the Desulfovibrionacea and Geobactereacea families play crucial roles in the bioenergetics of these microorganisms. Thermodynamic studies using NMR and visible spectroscopic techniques on tetraheme cytochromes c(3) isolated from Desulfovibrio spp. and more recently on a triheme cytochrome from Geobacter sulfurreducens showed that the properties of each redox centre are modulated by the neighbouring redox centres enabling these proteins to perform energy transduction and thus contributing to cellular energy conservation. Electron/proton transfer coupling relies on redox-linked conformational changes that were addressed for some multiheme cytochromes from the comparison of protein structure of fully reduced and fully oxidised forms. In this work, we identify for the first time in a multiheme cytochrome the simultaneous presence of two different conformations in solution. This was achieved by probing the different oxidation stages of a triheme cytochrome isolated from G. sulfurreducens using 2D-NMR techniques. The results presented here will be the foundations to evaluate the modulation of the redox centres properties by conformational changes that occur during the reoxidation of a multiheme protein.


Assuntos
Citocromos c/química , Citocromos c/ultraestrutura , Geobacter/enzimologia , Heme/química , Oxigênio/química , Oxirredução , Conformação Proteica
15.
Biophys J ; 93(8): 2756-66, 2007 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-17557782

RESUMO

The infrared (IR) absorption of the amide I band for the loop structure may overlap with that of the alpha-helices, which can lead to the misassignment of the protein secondary structures. A resolution-enhanced Fourier transform infrared (FTIR) spectroscopic method and temperature-jump (T-jump) time-resolved IR absorbance difference spectra were used to identify one specific loop absorption from the helical IR absorption bands of horse heart cytochrome c in D2O at a pD around 7.0. This small loop consists of residues 70-85 with Met-80 binding to the heme Fe(III). The FTIR spectra in amide I' region indicate that the loop and the helical absorption bands overlap at 1653 cm(-1) at room temperature. Thermal titration of the amide I' intensity at 1653 cm(-1) reveals that a transition in loop structural change occurs at lower temperature (Tm=45 degrees C), well before the global unfolding of the secondary structure (Tm approximately 82 degrees C). This loop structural change is assigned as being triggered by the Met-80 deligation from the heme Fe(III). T-jump time-resolved IR absorbance difference spectra reveal that a T-jump from 25 degrees C to 35 degrees C breaks the Fe-S bond between the Met-80 and the iron reversibly, which leads to a loop (1653 cm(-1), overlap with the helical absorption) to random coil (1645 cm(-1)) transition. The observed unfolding rate constant interpreted as the intrachain diffusion rate for this 16 residue loop was approximately 3.6x10(6) s(-1).


Assuntos
Citocromos c/química , Citocromos c/ultraestrutura , Modelos Químicos , Modelos Moleculares , Espectrofotometria Infravermelho/métodos , Simulação por Computador , Difusão , Cinética , Movimento (Física) , Conformação Proteica
16.
Biosens Bioelectron ; 23(2): 241-7, 2007 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-17532618

RESUMO

A method for the electrochemical detection of superoxide radical was developed, based on cytochrome c (cyt c) immobilized on the binary self-assembled monolayers (SAMs) of thioctic acid (T-COOH) and thioctic amide (T-NH2) on gold electrode. The sensor works by electrochemically detecting cyt c reduced by the superoxide radical generated by a xanthine-XOD system. The electrochemical properties of immobilized cyt c were investigated in aqueous buffer and in a mixture of aqueous and organic solvents. The interaction of superoxide radical with the modified electrode was characterized in phosphate buffer solution (PBS) and in the mixtures of both PBS and dimethyl sulfoxide (DMSO) and PBS and glycerol (Gly). The results showed that the sensors responded immediately to superoxide radical in PBS and gave a steady-state anodic current within 10s during the generation of superoxide radical. In 40% DMSO and in 30% Gly solution, the current response reached a steady-state anodic current within 20s. The sensor could also be used to estimate superoxide dismutase (SOD).


Assuntos
Técnicas Biossensoriais/instrumentação , Citocromos c/química , Eletroquímica/instrumentação , Compostos Orgânicos/química , Superóxidos/análise , Água/química , Técnicas Biossensoriais/métodos , Materiais Revestidos Biocompatíveis/química , Misturas Complexas/química , Citocromos c/ultraestrutura , Eletroquímica/métodos , Enzimas Imobilizadas/química , Desenho de Equipamento , Análise de Falha de Equipamento , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Solventes/química , Propriedades de Superfície
17.
Biochemistry ; 45(45): 13447-53, 2006 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-17087498

RESUMO

Recent publications described the formation of millimeter-length fibers by diverse lipid-binding proteins (e.g., histone H1, cytochrome c, indolicidin, and endostatin) when they are mixed with 80:20 phosphatidylcholine/phosphatidylserine vesicles. Further, these fibers displayed amyloid characteristics when stained with Congo Red. In the study presented here, we found by FTIR the amide I absorption band to reveal significant variation in fibers formed by cytochrome c, with some consisting of cytochrome c in a nativelike conformation and some exhibiting strong amyloid (beta-sheet) characteristics. Protein structure also varied from amyloid to nearly native within single fibers. Fibers were frequently blue or bluish and sometimes iridescent, likely due to interference of light in the fibers. The amyloid-type amide I band was observed for blue fibers only. AFM shows that fibers consist of smaller 3-4 nm diameter fibers with 10 nm lateral spacing.


Assuntos
Citocromos c/química , Citocromos c/ultraestrutura , Fosfolipídeos/farmacologia , Amiloide/química , Animais , Cor , Cavalos , Lipossomos , Microscopia de Força Atômica , Conformação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier
18.
Biophys J ; 91(9): 3415-24, 2006 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16891374

RESUMO

Protein molecules typically unfold (denature) when subjected to extremes of heat, cold, pH, solvent composition, or mechanical stress. One might expect that shearing forces induced by a nonuniform fluid flow would also destabilize proteins, as when a protein solution flows rapidly through a narrow channel. However, although the protein literature contains many references to shear denaturation, we find little quantitative evidence for the phenomenon. We have investigated whether a high shear can destabilize a small globular protein to any measurable extent. We study a protein (horse cytochrome c, 104 amino acids) whose fluorescence increases sharply upon unfolding. By forcing the sample through a silica capillary (inner diameter 150-180 microm) at speeds approaching 10 m/s, we subject the protein to shear rates dv(z)/dr as large as approximately 2 x 10(5) s(-1) while illuminating it with an ultraviolet laser. We can readily detect fluorescence changes of <1%, corresponding to shifts of < approximately 0.01 kJ/mol in the stability of the folded state. We find no evidence that even our highest shear rates significantly destabilize the folded protein. A simple model suggests that extraordinary shear rates, approximately 10(7) s(-1), would be required to denature typical small, globular proteins in water.


Assuntos
Citocromos c/química , Citocromos c/ultraestrutura , Microfluídica/métodos , Modelos Químicos , Modelos Moleculares , Animais , Simulação por Computador , Cavalos , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Resistência ao Cisalhamento , Estresse Mecânico
19.
J Am Soc Mass Spectrom ; 16(9): 1493-1497, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16019223

RESUMO

The folding pathways of gas-phase cytochrome c ions produced by electrospray ionization have been studied by an ion trapping/ion mobility technique that allows conformations to be examined over extended timescales (10 ms to 10 s). The results show that the +9 charge state emerges from solution as a compact structure and then rapidly unfolds into several substantially more open structures, a transition that requires 30-60 ms; over substantially longer timescales (250 ms to 10 s) elongated states appear to refold into an array of folded structures. The new folded states are less compact than those that are apparent during the initial unfolding. Apparently, unfolding to highly open conformations is a key step that must occur before +9 ions can sample more compact states that are stable at longer times.


Assuntos
Citocromos c/química , Citocromos c/ultraestrutura , Gases/química , Espectrometria de Massas por Ionização por Electrospray/instrumentação , Espectrometria de Massas por Ionização por Electrospray/métodos , Cinética , Transição de Fase , Conformação Proteica , Dobramento de Proteína , Renaturação Proteica
20.
Biosens Bioelectron ; 20(9): 1878-83, 2005 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-15681209

RESUMO

Molecularly imprinted thin films consisting of proteins embedded in polymerised aminophenyl boronic acid have been made on glass supports. The protein contents of the films have been optimised to achieve a maximum energy of interaction between the film and the native template. The fabrication of the films and the subsequent removal from their surfaces of the imprint proteins has been shown to be a facile and easily reproduced process. The enthalpy changes associated with the rebinding of the films with their original templates (lysozyme and cytochrome c) and with non-native templates has been examined by micro-calorimetry. The results demonstrate that thin films can be successfully imprinted as shown by the significant reduction in the enthalpy (DeltaH) observed when the films were rebound with proteins other than the original templates. Additionally, it was shown that after binding, non-template proteins could be removed by washing and a greater enthalpy again observed when the films were rebound with the native protein compared to that which had been found with the non-native protein.


Assuntos
Técnicas Biossensoriais/métodos , Materiais Revestidos Biocompatíveis/química , Citocromos c/química , Citocromos c/ultraestrutura , Muramidase/química , Muramidase/ultraestrutura , Sítios de Ligação , Técnicas Biossensoriais/instrumentação , Materiais Revestidos Biocompatíveis/análise , Transferência de Energia , Enzimas Imobilizadas/análise , Enzimas Imobilizadas/química , Teste de Materiais , Membranas Artificiais , Ligação Proteica , Propriedades de Superfície
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